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Abstract:

A fermented liquid feed composition for a target animal is provided, the
feed composition being prepared by the fermentation of a feed substrate
with a lactic acid producing bacteria, the lactic acid bacteria being
characterised by: a) being viable under the conditions prevailing in
the gastrointestinal tract of the target animal; b) being an aggregating
bacteria and/or co-aggregating with one or more pathogens; and c) being
able to produce upon fermentation in the feed substrate lactic acid in an
amount of at least a minimum inhibitory concentration of lactic acid.
A method of producing a fermented liquid feed composition is also
provided. Preferred lactic acid producing bacteria for use in the
composition and method are Lactobacillus.

Claims:

1-39. (canceled)

40. A fermented liquid feed composition for a target animal, the feed
composition being prepared by the fermentation of a feed substrate with a
lactic acid producing bacteria, the lactic acid bacteria being
characterised by: a) being viable under the conditions prevailing in the
gastrointestinal tract of the target animal; b) being a bacteria capable
of aggregating and/or co-aggregating with one or more pathogens; and c)
being able to produce upon fermentation in the feed substrate lactic acid
in an amount of at least a minimum inhibitory concentration of lactic
acid.

41. The fermented liquid feed composition according to claim 40, wherein
the target animal is poultry or mammals.

42. The fermented liquid feed composition according to claim 41, wherein
the target animal is chickens or pigs.

43. The fermented liquid feed composition according to claim 40, wherein
the substrate is selected from the group consisting of plants, plant
material, and organic residue.

44. The fermented liquid feed composition according to claim 43, wherein
the plant material is selected from the group consisting of grass;
cereals and grains, such as wheat, barley, maize, rice, sorghum and rye;
root crops, such as potatoes, swedes, fodder beet, sugar beet and the
like; pulses and seeds, such as beans, peas, soya bean rapeseed;
brassiccas and the like.

45. The fermented liquid feed composition according to claim 43, wherein
the organic residue comprises co-products or remnants from the group
consisting of dairy operations, such as whey, curd, skimmed milk, ice
cream, yoghurt, butter and cheese; the baking and confectionary industry;
the beverage industry; brewing and distilling; the extraction of cooking
oil; meat and fish slaughter and processing; and the production of
bio-fuels.

46. The fermented liquid feed composition according to claim 40, wherein
the feed substrate has a water content of at least 20% by weight.

47. The fermented liquid feed composition according to claim 40, wherein
the ratio of dry feed substrate to water are from 1:0.25 to 1:4.

48. The fermented liquid feed composition according to claim 40, wherein
the lactic acid producing bacteria are viable under a plurality of
conditions of pH corresponding to a plurality of locations in the
gastrointestinal tract of the target animal.

49. The fermented liquid feed composition according to claim 40, wherein
the lactic acid producing bacteria are proven viable in an in vitro
experiment modelling the conditions in the gastrointestinal tract of the
target animal.

50. The fermented liquid feed composition according to claim 49, wherein
the in vitro experiment employs the feed substrate as the growth medium
for the lactic acid producing bacteria.

52. The fermented liquid feed composition according to claim 51, wherein
the lactic acid producing bacteria are coaggregating with respect to
bacteria that are harmful or pathogens to the target animal.

54. The fermented liquid feed composition according to claim 40, wherein
the lactic acid producing bacteria are capable of producing at least 200
mMol of lactic acid in 48 hours upon fermentation at 30.degree. C. in a
growth medium consisting of MRS broth with 2% by weight glucose.

55. The fermented liquid feed composition according to claim 54, wherein
the lactic acid producing bacteria produces at least 250 mMols of lactic
acid under the said conditions.

56. The fermented liquid feed composition according to claim 40, having a
pH of 4.5 or lower.

58. The fermented liquid feed composition according to claim 40, wherein
the lactic acid producing bacteria is antagonistic against one or more
pathogens common in the target animal.

59. The fermented liquid feed composition according to claim 40, wherein
the concentration of lactic acid producing bacteria in the fermented feed
is at least 10.sup.6 CFU/ml.

60. The fermented liquid feed composition according to claim 40, wherein
the lactic acid producing bacteria comprise strains of bacteria selected
from the group consisting of Lactobacillus and Pediococcus.

61. The fermented liquid feed composition according to claim 60, wherein
the lactic acid producing bacteria comprises a strain of Lactobacillus
selected from the species of Lactobacillus plantarum and Lactobacillus
salivarius.

63. An inoculant for the preparation of a fermented feed from a feed
substrate, the inoculant comprising a viable culture of a lactic acid
producing bacteria having the following characteristics: a) being viable
under the conditions prevailing in the gastrointestinal tract of the
target animal; b) being an aggregating bacteria and/or co-aggregating
with one or more pathogens; and c) being able to produce upon
fermentation in the feed substrate lactic acid in an amount of at least a
minimum inhibitory concentration of lactic acid.

64. The inoculant according to claim 63, wherein the concentration of
lactic acid producing bacteria in the inoculant is from 10.sup.5 to
10.sup.9 CFU/ml.

65. The inoculant according to claim 63, wherein the lactic acid
producing bacteria comprise strains of bacteria selected from the group
consisting of Lactobacillus and Pediococcus.

66. The inoculant according to claim 65, wherein the lactic acid
producing bacteria comprises a strain of Lactobacillus selected from the
species of Lactobacillus plantarum and Lactobacillus salivarius.

68. A method for preparing a fermented feed composition, the method
comprising fermenting a feed substrate with a lactic acid producing
bacteria, the lactic acid bacteria being characterised by: a) being
viable under the conditions prevailing in the gastrointestinal tract of
the target animal; b) being an aggregating bacteria and/or co-aggregating
with one or more pathogens; and c) being able to produce upon
fermentation in the feed substrate lactic acid in an amount of at least a
minimum inhibitory concentration of lactic acid.

69. The method according to claim 68, wherein the lactic acid producing
bacteria comprise strains of bacteria selected from the group consisting
of Lactobacillus and/or Pediococcus.

70. The method according to claim 69, wherein the lactic acid producing
bacteria comprises a strain of Lactobacillus selected from the species of
Lactobacillus plantarum and Lactobacillus salivarius.

73. A method for improving the general health of a target animal, the
method comprising administering to the animal lactic acid producing
bacteria having the following characteristics: a) being viable under the
conditions prevailing in the gastrointestinal tract of the target animal;
b) being an aggregating bacteria and/or co-aggregating with one or more
pathogens; and c) being able to produce upon fermentation in the feed
substrate lactic acid in an amount of at least a minimum inhibitory
concentration of lactic acid.

74. The method according to claim 73, wherein the lactic acid producing
bacteria are administered by means selected from the group consisting of
the water provided to the animal and the animal feed.

Description:

[0001] The present invention relates to feed materials for animals, to the
provision of fermented animal feed products, methods for their
preparation and the use thereof.

[0002] It is common practice to prepare animal feeds as dry compositions,
that is up to 15% moisture, to avoid spoilage. The dry feed material may
be stored for extended periods of time and transported with little or no
degradation. However, the cost of preparing dried feed is increasing. In
particular, in excess of 60% of the energy costs on preparing the dry
feed are consumed in the actual drying stage. Accordingly, there is a
growing need for an alternative to the known dry animal feeds.

[0003] One such alternative are moist or liquid feeds. In this regard,
moist feeds, such as those fed to chickens and other poultry, contain up
to 30% by weight of water. Liquid feeds, such as those fed to pigs,
contain up to 70% by weight of water. Known for some time, liquid feeds
can be difficult to formulate, prepare and store in a cost effective
manner. In addition, moist or liquid feeds are difficult to store and
transport over long distances. In particular, wet or liquid animal feeds
are very prone to spoiling due to the growth of mould, bacteria and
yeast, making the long term storage of wet and liquid animal feeds a
difficult prospect. There is therefore a need to address the problem of
storing and transporting wet and liquid feeds. It would also be most
advantageous if the range of starting materials for preparing wet and
liquid animal feeds could be extended. Currently, co-products and
residues from industries such as dairy, bakery and distilling are used to
prepare wet and liquid feed for animals. Raw materials with significant
future potential are the co-products of bioethanal and biofuel
production. These are already incorporated into diets following drying,
which is an extremely energy demanding mode of treatment. It would be
beneficial, if a way can be found to formulate these co-products into a
moist or liquid feed. Other potential sources for raw materials for
incorporation into moist and liquid animal feeds include human food grade
residues from the slaughter process and from meat and fish processing.
The value of much of this material is currently lost due to poor storage,
drying or disposal to landfill. All these processes have a high energy
demand and adverse environmental impact.

[0004] One improvement to the preparation of wet and liquid animal feeds
is the inoculation of the feed raw material with one or more suitable
microorganisms, to produce a so-called `fermented feed`. This process is
synonymous with the process of `ensiling`, which is the ubiquitous method
used for the preservation of herbage for feeding to ruminant animals. The
inoculant is selected to inhibit the growth of mould, yeasts and spoilage
bacteria that will propagate in and spoil the feed material. The process
of producing fermented feed can be seen as a form of `biopreservation`.
EP 0 906 952 discloses a bacterial strain for the ensiling of straw
fodder. The strain, of the genus Lactococcus, was found to be effective
in inhibiting the growth or yeast, clostridia, mould, gram positive
bacteria and certain gram negative bacteria in the ensiling of green
fodder.

[0005] Further, US 2002/0054935 is concerned with a livestock feed
composition suitable for the fattening of livestock, such as cattle,
goats, sheep, swine and fowl. The nutritional value of the livestock feed
is increased by inoculation with one or more strains of Aspergillus. The
livestock feed treated in this way consists of a fibrous feed material, a
cereal, and an organic waste material. It appears that the Aspergillus is
used to modify the nutrient content of the feed. However, this can have
disadvantageous results, as many Aspergillus spp. produce mycotoxins
harmful to many animals.

[0006] U.S. Pat. No. 6,403,084 is concerned with mixed cultures for
improved fermentation and aerobic stability of silage. The problem of
aerobic instability of silage is addressed, in particular the rapid
growth of yeast and mould that can occur, resulting in the silage being
spoiled. Further, it is noted that silage may be spoiled by the growth of
yeast, even when inoculated and subjected to a good fermentation phase,
in which microorganisms are used to ferment the silage and produce lactic
acid, reducing the pH and giving rise to acid conditions. Acid-tolerant
yeasts are considered to be responsible for the spoilage of fermented
silage. As a solution to these problems, U.S. Pat. No. 6,403,084 proposes
inoculating the silage with a combination of the homofermentative lactic
acid bacteria Lactobacillus plantarum and the heterofermentative lactic
acid bacteria Lactobacillus buchneri or Lactobacillus brevis. The
aforementioned combination of microorganisms is alleged to provide
sufficiently low pH conditions to preserve the silage and prevent
spoiling due to the growth of mould and yeast.

[0007] WO 99/18188 describes a feed product for horses. The feed product
comprises one or more strains of Lactobacillus having the ability to
colonize the equine intestines. The microorganisms were isolated from the
gastric or intestinal mucosa of horses.

[0008] GB 2,167,639 discloses a process for the treatment of industrial or
agricultural waste matter, such as animal protein. The process involves
chopping the waste as an aqueous mass and treating the resulting material
with proteolytic enzymes to form a suspension, obtaining a gelatinised
starch content in the suspension and adding to the suspension amylolytic
enzymes and a lactic acid producing culture. The resulting mixture is
fermented to produce simple sugars and lactic acid.

[0009] U.S. Pat. No. 4,214,985 relates to sewerage treatment. The
treatment involves inoculating sewerage sludge with L. plantarum bacteria
and a carbohydrate, such as lactose. The resulting mixture is fermented
until the pH falls below 4.0. The thus produced composition is used as a
soil extender.

[0010] JP 2007082468 is concerned with providing a microorganisms
preparation for feed. The preparation comprises particular strains of
Lactobacillus plantarum and/or Bacillus subtilis. Fermented feeds may be
produced by adding the microorganisms to organic wastes, such as silage
grass, and fermenting.

[0011] WO 89/05849 describes the selection of lactic acid bacteria
isolated from the gastrointestinal tract of pigs for their ability to
survive in the environment of the gastrointestinal tract and to adhere to
the epithelium of the gastrointestinal tract of the target animal. The
bacteria selected with these properties may be included in a fermented
milk product for human consumption or in a veterinary composition for
providing to pigs for the prevention or treatment of gastrointestinal
diseases.

[0012] More recently, WO 2008/006382 discloses homofermented liquid animal
feed products. As discussed in WO 2008/006382, the production of
fermented animal feeds using microorganism-containing inoculants is very
difficult, often leading to the fermented feed containing pathogenic
bacteria, such as Vibrio spp., Campylobacter spp., Salmonella spp., E.
coli, and Staphylococcus aureus. The fermented feed may also contain a
high content of various yeasts and moulds. It is noted that the ingestion
by the livestock of such inappropriately fermented feeds may result in
morbidity and mortality. WO 2008/006382 notes that the sterile handling
of bacteria required by farmers wishing to prepare their own fermented
feed is often impossible to achieve. Further, there is a practice of
using a continuous fermentation process, in which a portion of one batch
of fermented feed is used as an inoculum for a subsequent fermentation
batch. This leads to a gradual build up of harmful and undesirable
microorganisms in the fermented feed. In an attempt to address these
problems, WO 2008/006382 proposes a method for preparing a fermented
mixed feed, the method comprising: providing a liquid fermented product;
providing a feed product to be fermented; combining the aforementioned
products; and fermenting the feed product using the liquid fermented
product as an inoculum. A fermented feed prepared by this method is also
described.

[0013] While proposals have been made to provide fermented feeds that are
resistant to spoilage due to the growth of yeasts, moulds, bacteria and
other organisms, there is still a need for an improved fermented feed
that may be stored for extended periods of time and transported, without
significant spoilage.

[0014] As mentioned in WO 2008/006382, a further issue relating to
feedstuffs, in particular moist or liquid feed materials, is the health
and wellbeing of the livestock consuming the feeds. As noted in WO
2008/006382, a poorly fermented feed may be a source of microorganisms
harmful to the animals consuming the feed. More generally, animals are
susceptible to a wide range of infections arising from microorganisms
that enter and colonise the gastrointestinal (GI) tract of the animal. EP
0 955 061 addresses the issue of gastroenteric infections in pigs, in
particular porcine rotavirus, porcine coronavirus, enterotoxigenic and
enteropathogenic strains of Escherichia coli, Clostridium sp., Salmonella
sp., Serpulina hyodysenteriae, Serpulina pilosicoli, Lawsonia
intracellularis, Isospora suis, and Cryptosporidium. As a solution to the
problem of gastroenteric infections in pigs, EP 0 955 061 proposes an
oral product characterised by containing at least one specific antibody
to the aforementioned microorganisms, derived from the egg yolks of
immunized hens. It is noted in EP 0 955 061 that lactacidogenic bacteria
administered to pigs can have a probiotic effect, suppressing the
propagation of the enteropathogenic or enterotoxigenic bacteria and
enhance the activity of the animal's immune system. Accordingly, a
preferred embodiment of EP 0 955 061 includes one or more lactic acid
bacteria, such as Enterococcus spp. and Lactobacillus spp.

[0015] As discussed in EP 0 955 061, young animals are particularly
susceptible to infections of the GI tract, leading to illness and death.
Ways of improving the health and wellbeing of finishing pigs are
described by P. Brooks et al., `The Effect on Biological Performance and
Faecal Microbiology of Feeding Finishing Pigs on Liquid Diets Fermented
with Lactic Acid Bacteria`, SafePork, 2005, page 149. Brooks et al. note
that fermented liquid feeds (FLF) have been shown to reduce the incidence
of salmonella in pigs. In particular, it was found that a lactic acid
concentration of 70 mMol/kg in the fermented feed exhibited
bacteriostatic activity with respect to Salmonella spp., but higher
concentrations of lactic acid in excess of 100 mMol/kg were needed to be
bactericidal. However, Brooks et al. had found that natural fermentations
had produced unpredictable results in commercial feed units and referred
to a study that found that only 3% of commercial fermentations of wheat
and barley produced more than 75 mMol/kg of lactic acid after 24 hours of
fermentation. It was concluded that fermentations to produce lactic acid
in high concentrations relying on indigenous microorganisms present in
the grains could not be relied upon for commercial production of
fermented feeds. Brooks et al. conducted experiments using specific LAB
to examine the effect on biological performance and faecal microbiology
of pigs fed diets of fermented liquid feeds. The results showed that the
pigs retained good health when fed on the fermented liquid feed, showing
no change in average daily weight gain when fed with the FLF compared
with a standard feed. In addition, the experiments showed that, while the
fermented feed contained lactic acid bacteria in high concentrations, the
concentration of LAB in the faeces of the pigs remained unchanged.
However, analysis of the faeces for the presence of coliforms indicated
that the coliform content was reduced in the pigs fed with the FLF diet.
This in turn indicated an improvement in the health of the pig and a
lower risk of infection and illness. It was concluded that the selection
of the LAB used for fermentation was important in achieving the reduction
in coliforms.

[0016] As noted by Brooks et al., achieving a specific concentration of
lactic acid in the fermented feed is important in achieving the
beneficial effects of the fermented feeds. Techniques for measuring the
concentrations of lactic acid in fermented feeds are described by S. J.
Niven, et al., The Simultaneous Determination of Short Chain Fatty Acid,
Monosaccharides and Ethanol in Fermented Liquid Pig Diets', Animal Feed
Science and Technology, 117 (2004), pages 339 to 345.

[0017] The thesis of V. Demeckova, `Benefits of Fermented Liquid Diets for
Sows and their Piglets`, Department of Agriculture and Food, Faculty of
Land, Food and Leisure, University of Plymouth, July 2003, describes
experiments conducted with liquid feed fermented with Lactobacillus
plantarum to determine their effects on the antimicrobial and potential
immunological effects on sows in late gestation periods. The results
indicated that certain strains of Lactobacillus were both an effective
inoculant for the preparation of fermented liquid feeds, as well as
providing probiotic activity to the sow once the fermented feed was
ingested. Significantly, immunoglobulin levels in the sows' colostrums
were increased. Colostrum from sows fed fermented feed also increased the
mitogenic activity of blood lymphocytes and enterocytes.

[0018] These factors could in turn improve the resistance of the sows and
their piglets to pathogen challenges.

[0019] Drago, L., et al., `Inhibition of in vitro growth of
enteropathogens by new Lactobacillus isolates of human intestinal
origin`, FEMS Microbiology Letters, 153 (1997), pages 455 to 463,
describe experiments to isolate strains of Lactobacillus from the faeces
of new born infant humans and examine their effect in co-cultures on
certain pathogenic bacteria. The experiments conducted were entirely in
vitro and, while showing some beneficial effects of the Lactobacillus
strains in reducing the growth of certain pathogens, did not relate at
all to the formulation of feeds for animals.

[0020] It would be most advantageous if a fermented liquid feed
composition could be provided that may be prepared on a commercial scale
from a wide range of raw and starting materials, that would be
biopreserved and exclude potentially harmful enteropathogens. It would be
further advantageous if the fermented feed could provide a probiotic
effect to the animals receiving it and reduce or prevent illness of the
animals due to infections and pathogenic challenge.

[0021] The inventors have now found that such a fermented liquid feed
composition may be produced using one or more lactic acid bacteria
possessing certain characteristics.

[0022] Accordingly, in a first aspect, the present invention provides a
fermented liquid feed composition for a target animal, the feed
composition being prepared by the fermentation of a feed substrate with a
lactic acid producing bacteria, the lactic acid bacteria being
characterised by: [0023] a) being viable under the conditions
prevailing in the gastrointestinal tract of the target animal; [0024] b)
being a bacteria capable of aggregating and/or coaggregating with one or
more pathogens; and [0025] c) being able to produce upon fermentation in
the feed substrate lactic acid in an amount of at least a minimum
inhibitory concentration of lactic acid.

[0026] It has been found that a fermented feed according to the present
invention produced by the fermentation of a feed substrate with a lactic
acid producing bacteria having the characteristics set out above provides
significant advantages over known feeds. In particular, the feed is able
to be stored for extended periods of time and be transported without
spoiling, the fermented feed being resistant to the growth of mould and
yeasts and resistant to invasion and colonisation by bacteria potentially
harmful to the target animals and other livestock. Further, the fermented
feed, once consumed, provides significant protection for the animals
against infection by bacteria, in particular pathogenic bacteria likely
to cause serious illness or death of the animal. The fermented feed of
the present invention achieves this by enhancing the barrier function of
the upper gastrointestinal tract of the target animal. This advantage is
particularly significant in the feeding of newly born and newly weaned
animals, and newly hatched birds. In the large scale rearing of animals,
for example cattle, pigs and poultry, there are particular challenges
when young animals are removed from their mothers (weaned) and reared in
a different environment. The weaning process removes immunoglobulin
support provided by the mother's milk and precipitates changes to the gut
ecosystem. This, in turn, leaves the young animals open to infection with
a wide range of potentially harmful microorganisms. The high mortality
rate of young animals is at least in part due to animals succumbing to
such infections of microorganisms. The fermented feed of the present
invention reduces or eliminates this risk, by populating the
gastrointestinal tract of the young animal with healthy, beneficial
microorganisms, in turn providing protection of the young animals against
infection by pathogenic microorganisms.

[0027] The fermented feed of the present invention is provided for a
target animal, which may determine such factors as the composition of the
feed and the particular bacteria employed in the fermentation of the feed
substrate. The fermented feed of the present invention may be provided
for a wide range of animals and livestock, including mammals and poultry.
Examples of target mammals include all the mammals farmed or reared,
including horses, sheep, goats, pigs, cattle and deer, as well as animals
reared for fur, such as mink and the like. Examples of target poultry
include all the birds reared and farmed on a commercial scale, including
chickens, ducks, geese, quail and turkeys, as well as game birds, such as
pheasants, partridges and the like. The fermented feed may also be
provided to farmed and ornamental fish and crustaceans. Further, the
fermented feed may be provided for animals kept as domestic pets, such as
dogs, cats, rabbits and the like.

[0028] In one preferred embodiment, the fermented feed of the present
invention is advantageously formulated for providing to poultry,
including chickens, quail, turkeys, geese, ducks and the like.

[0029] In a second preferred embodiment, the fermented feed of the present
invention is advantageously formulated for providing to mammals, in
particular pigs and ruminants, such as cattle and sheep, particularly
during the post-natal and pre-ruminant stage and veal calves, in which
ruminant function my be delayed.

[0030] The fermented feed of the present invention may be provided to any
age of target animal, from newly born animals or newly hatched birds to
mature adult animals. The fermented feed has been found to be
particularly advantageous when provided to newly born and newly weaned
animals or newly hatched birss, where the fermented feed provides such
advantages as increased weight gain of the young animals and a reduction
in infection with potentially harmful or pathogenic microorganisms. This
results in a reduction in the harmful or pathogenic microorganisms shed
by the animals, in turn increasing the health of other animals being
reared in the same environment and the ultimate consumer of the animal
and/or its products.

[0031] The fermented feed is prepared from a feed substrate, which is
inoculated with a culture containing the lactic acid producing bacteria
and fermented. The thus inoculated and fermented substrate may itself
form the finished feed. Alternatively, the substrate, once fermented may
be added to other feed materials, in order to provide the other materials
with the biopreservative effects.

[0032] The feed substrate may be any suitable substrate that may be
consumed by the target animals in a fermented condition. The feed
substrate may consist of a substrate from a single or source or,
alternatively may comprise a combination of substrates.

[0033] Suitable substrates include organic materials, such as plants or
plant material, for example, fibrous plant material, such as grass;
cereals and grains, such as wheat, barley, maize, rice, sorghum and rye;
whole crop cereals, maize silage and corn cob meal; root crops, such as
potatoes, swedes, fodder beet, sugar beet and the like; pulses and seeds,
such as beans, peas, soya bean and rapeseed (and their residues);
brassiccas and the like. Further substrates include organic residues,
such as materials produced as co-products or residues from dairy
operations, such as whey, curd and skimmed milk, ice cream, yoghurt,
off-specification butter and cheese; from the baking and confectionary
industry, such as biscuit meals, cereal residues, misshapen and
off-specification breads, cakes and biscuits; from the beverage industry,
such as fruit pulps, grape pulp, coffee and chocolate residues; brewing
and distilling residues; from cooking oil extraction, such as rapeseed
meal, soya bean meal and olive pulp; from meat and fish slaughter and
processing; or the production of bio-fuels.

[0034] To be suitable for fermentation, the feed substrate should contain
water in an amount sufficient to support fermentation with the lactic
acid producing bacteria. Preferably, the feed substrate has a water
content of at least 20% by weight, more preferably at least 30% by
weight, still more preferably at least 40% by weight. Preferred ratios of
dry feed substrate to water are dependant on the target species to be fed
and range from 1:0.25 to 1:4, more preferably from 1:0.4 to 1:2. One
preferred ratio of dry feed substrate to water is about 1:1.2 for
chickens and 1:2.5 for pigs. The precise water content of the feed
substrate will be determined by such factors as the nature and
composition of the feed substrate, the lactic acid producing bacteria
being employed and the end use of the fermented feed and target animal.
References herein to a `moist feed` or `liquid feed` are to a feed
material containing at least the minimum water content to support
fermentation of the feed by the lactic acid producing bacteria and the
terms `moist feed` and `liquid feed` are to be understood and interpreted
accordingly.

[0035] The feed substrate may contain sufficient water to support
fermentation with the lactic acid bacteria. If not, water should be added
to the feed substrate to achieve the water content required for
fermentation.

[0036] To produce the fermented feed of the present invention, the feed
substrate is inoculated with lactic acid producing bacteria and
fermented. The lactic acid producing bacteria employed in the present
invention are characterised by the following features: [0037] a) The
bacteria are viable under the conditions prevailing in the
gastrointestinal tract of the target animal; [0038] b) The bacteria are
capable of aggregating and/or co-aggregating with one or more pathogens;
and [0039] c) The bacteria are capable of producing lactic acid upon
fermentation with the feed substrate to at least a minimum inhibitory
concentration in the fermented feed.

[0040] Bacteria having the three characteristics (a) to (c) give rise to
the advantageous properties of the fermented feed of the present
invention. As noted above, the feed substrate is inoculated with the
lactic acid producing bacteria.

[0041] Accordingly, in a further aspect, the present invention provides an
inoculant for the preparation of a fermented feed from a feed substrate,
the inoculant comprising a viable culture of a lactic acid producing
bacteria having the following characteristics: [0042] a) being viable
under the conditions prevailing in the gastrointestinal tract of the
target animal; [0043] b) being a bacteria capable of aggregating and/or
co-aggregating with one or more pathogens; and [0044] c) being able to
produce upon fermentation in the feed substrate lactic acid in an amount
of at least a minimum inhibitory concentration of lactic acid.

[0045] The inoculant may be in any suitable form and of any suitable
composition so as to contain viable lactic acid producing bacteria for
populating and fermenting the feed substrate. Preferred presentations for
the inoculant are freeze dried or as a liquid culture.

[0046] The inoculant should contain the lactic acid producing bacteria in
a viable form and in sufficient concentration to allow the feed
substrate, once inoculated, to ferment and produce the required number of
viable lactic acid producing bacteria and the required concentration of
lactic acid in the fermented feed. A typical number of lactic acid
producing bacteria in the inoculant is from 105 to 109 CFU/ml,
more preferably about 106 CFU/ml, if presented in liquid form or
105 to 109 CFU/g, more preferably about 106 CFU/g if
presented in freeze dried form.

[0047] For example, a suitable inoculant for a substrate is 0.1% of a
liquid broth culture containing 109 CFU/ml of the lactic acid
producing bacteria or 0.1% of a freeze dried culture containing 109
CFU/g of the lactic acid producing bacteria.

[0048] The inoculant organism may be presented in a suitable carrier to
maintain shelf life and facilitate accurate dispersion when added to the
substrate. Such methods are known in the art and readily understood by
the person skilled in the art.

[0049] As a first characteristic, the lactic acid producing bacteria
should be viable and survive in the gastrointestinal tract of the target
animal. The conditions in the gastrointestinal tract of many animals are
severe enough to prevent the colonisation and growth of many
microorganisms. In particular, the upper gastrointestinal tract of many
animals is sufficiently acidic to prevent many species of microorganisms
from thriving and remaining viable. In order to provide the advantageous
properties of the fermented feed of the present invention, the lactic
acid producing bacteria used to ferment the feeds substrate should be
viable under the acidic conditions prevailing in the upper
gastrointestinal tract of the target animal and the alkaline conditions
encountered in the duodenum, and should remain viable in both the small
intestines and the large intestines. In addition, in the case of
fermented feed intended for providing to poultry, the lactic acid
producing bacteria should also remain viable under the conditions
prevailing in the crop and proventiculus, as well as the gizzard.

[0050] The viability of the bacteria in the gastrointestinal tract may be
determined by methods and techniques known in the art. In particular, the
microbial count of the viable lactic acid bacteria in the faeces of
target animals fed a diet containing viable lactic acid bacteria may be
measured. Alternatively, the lactic acid bacteria count in the
gastrointestinal tract of poultry fed a diet containing the bacteria may
be determined using cloacal swabs. Such methods are known in the art and
readily understood by the person skilled in the art.

[0051] As a further alternative or in addition thereto, the viability of
the lactic acid producing bacteria in the gastrointestinal tract of the
target animal may be determined in vitro, in particular by measuring the
growth of the microorganisms under acidic conditions similar to or the
same as those prevailing in the upper gastrointestinal tract of the
target animal. Thus, in the case of a fermented feed intended for pigs,
the viability of the lactic acid producing bacteria may be determined
after exposure to pH 2 for 2 hours followed by buffering to pH 6.8 and
exposure to bile salts for 4 hours in a suitable feed substrate, to
represent conditions in the stomach and the small intestine of the target
animal. The acidity of the large intestine is generally similar to that
of the small intestine, meaning that viability in the large intestine is
most likely for all microorganisms surviving under conditions of lower pH
prevailing in the stomach. Similarly, viability in the gastrointestinal
tract of poultry may be determined by sequentially exposing the lactic
acid producing bacteria in a suitable feed substrate to a pH of from 4.4
to 4.5, as encountered in the crop and proventiculus, a pH of about 2.6,
as encountered in the gizzard, and to a pH of 6.2 in the presence of bile
salts as encountered in the small intestine of the target birds. Again,
conditions in the caecum are unlikely to adversely affect organisms that
survive the gizzard and small intestine.

[0052] In order to more accurately model the conditions of the
gastrointestinal tract of the target animal, it is further preferred that
the in vitro experiments described hereinbefore are conducted using the
feed substrate of the eventual fermented feed as the growth medium for
the microorganisms. In this way, any effects produced within the
gastrointestinal tract of the target animal when fed with the fermented
feed that may alter the conditions therein and/or the viability of the
lactic acid producing bacteria may be determined.

[0053] A procedure for the in vitro determination of the viability of a
lactic acid producing bacteria in the gastrointestinal tract of a target
animal is described in detail in Example 1 hereafter.

[0054] In a particularly preferred embodiment, the fermented feed of the
present invention comprises lactic acid producing bacteria that have been
demonstrated to be viable in the gastrointestinal tract of the target
animal using the aforementioned in vitro procedure employing the feed
substrate as growth medium for the microorganisms.

[0055] As a second requirement, the lactic acid producing bacteria of the
fermented feed of the present invention are aggregating bacteria, that is
the bacteria form aggregates. In addition, or alternatively, the bacteria
are capable of co-aggregating with other microorganisms, in particular
microorganisms that are pathogenic to the target animal, that is form
aggregates together with the other microorganisms. Preferably, the lactic
acid producing bacteria are both aggregating and co-aggregating. The
ability to aggregate and/or co-aggregate may be exhibited by the lactic
acid producing bacteria under the conditions in the feed substrate during
and after fermentation and/or under the conditions prevailing in the
gastrointestinal tract of the target animal. Preferably, the bacteria are
aggregating and/or co-aggregating both in the feed substrate and in the
gastrointestinal tract of the target animal.

[0056] The ability of the microorganisms to aggregate in vitro gives a
strong indication of their ability to adhere to the mucus layer in the
gut and the epithelial cells of the intestinal wall of the target animal
and, generally, to colonise the gastrointestinal tract. This in turn
increases the resistance of the target animal to infection by exclusion
of harmful or pathogenic microorganisms from attachment sites. Further,
the lactic acid producing bacteria are preferably ones that are
coaggregating, that is form coaggregations with other microorganisms, in
particular harmful or pathogenic bacteria. In particular, it is preferred
that the lactic acid producing bacteria are coaggregating with strains of
Salmonella, E. Coli, and/or Clostridium. This in turn increases the
passage and clearance of the harmful bacteria from the gut lumen.

[0057] The ability of a lactic acid producing bacteria to aggregate may be
determined by in vitro methods and techniques known in the art, for
example as described in Drago, L. et al., noted above. In particular, the
bacteria may be cultured in a suitable liquid growth medium, such as
Man-Rogosa-Sharpe (MRS) broth (available commercially). Bacterial
aggregates may be identified as grains or particles that develop in the
liquid culture medium, typically collecting at the bottom of the culture
vessel under the action of gravity and leaving a clear supernatant
liquid.

[0058] Similarly, the ability of the lactic acid producing bacteria to
coaggregate with other bacteria may be determined by preparing a
co-culture of the lactic acid producing bacteria with one or more target
bacteria in like manner with the formation of aggregates being observed
as grain-like particles that tend to settle in the culture, again leaving
a clear supernatant liquid.

[0059] While the formation of aggregates and coaggregates in the bacterial
cultures may be observed using the naked eye, as described above, further
and more detailed information regarding the aggregating ability of the
microorganisms may be obtained by using microscopy techniques, including
scanning electron microscopy (SEM).

[0060] A procedure for the identification of lactic acid bacteria that are
aggregating and coaggregating is set out in Example 2 below.

[0061] As a third characteristic, the lactic acid producing bacteria of
the fermented feeds of the present invention are capable of producing at
least a minimum inhibitory lactic acid concentration in the fermented
feed. In respect of the fermented feeds of the present invention, the
term `minimum inhibitory lactic acid concentration` is a reference to a
lactic acid producing bacteria that is capable of producing at least 150
mMol of lactic acid in 24 hours upon fermentation at 30° C. in a
growth medium consisting of MRS broth containing 2% by weight glucose. It
has been found that lactic acid producing bacteria that are capable of
producing this minimum concentration of lactic acid in the aforementioned
test are particularly advantageous in the preparation of fermented feeds
and providing significant health benefits to the target animals provided
with the feed.

[0062] The concentration of lactic acid in the culture medium may be
determined using methods known in the art, for example the method of
Niven, S. J., et al., The simultaneous determination of short chain fatty
acid monosaccharides and ethanol in fermented liquid pig diets', Animal
Feed Science and Technology, 117 (2004), (3-4), pages 339 to 345.

[0063] A procedure for identifying lactic acid producing bacteria capable
of producing at least the minimum inhibitory lactic acid concentration is
set out in Example 3 below.

[0064] More preferably, the lactic acid producing bacteria is capable of
producing at least 200 mMols of lactic acid under the aforementioned
procedure and test conditions, still more preferably at least 250 mMols
of lactic acid. Lactic acid concentrations of at least 300 mMols, more
preferably at least 350 mMols produced under the aforementioned test
conditions may also advantageously be applied.

[0065] In general, a higher concentration of lactic acid in the fermented
feed, and consequently a lower pH value for the fermented feed is to be
preferred. Accordingly, preferably the pH value of the fermented feed is
4.5 or lower, more preferably 4.0 or lower, still more preferably 3.5.
The lower limit of pH value and, hence, the upper limit for lactic acid
concentration will be, at least in part, determined by the target animal
and its ability and willingness to eat the fermented feed. As the pH is
lowered further, the target animals may refuse to eat the feed.

[0066] The lactic acid producing bacteria employed to prepare the
fermented feed of the present invention may be either homofermenting or
heterofermenting. Heterofermenting bacteria produce lactic acid as a
product of their metabolism, along with other organic acids, such as, for
example, acetic acid, propionic acid and butyric acid. However, it has
been found that the presence of significant quantities of these other
acid metabolites may adversely affect the taste of the fermented feed
and/or reduce the nutritional value of the feed to the target animal. In
contrast, homofermenting lactic acid producing bacteria are ones that
metabolise the feed substrate to produce lactic acid as the only acid
metabolite. Accordingly, it is preferred that the lactic acid producing
bacteria present in the fermented feed are homofermenting.

[0067] Further, the lactic acid producing bacteria used in the fermented
feed of the present invention are preferably antagonistic towards
pathogens common to the target animal. For example, in the case of
fermented feed intended to be provided to poultry, it is preferred that
the lactic acid producing bacteria have antagonistic activity against one
or more strains of Salmonella, Clostridium and E. coli.

[0068] A procedure for determining the antagonistic activity of a lactic
acid producing bacteria is set out in Example 4 below.

[0069] In addition, the lactic acid producing bacteria used in the
fermented feed of the present invention are preferably capable of
adhering to the epithelial cells of the gastrointestinal tract of the
target animal. In vitro methods for determining the adhesion of bacteria
in this manner are known in the art.

[0070] A procedure for determining the ability of the lactic acid
producing bacteria to adhere to the epithelial cells of the target animal
is set out in Example 5 below.

[0071] Suitable lactic acid producing bacteria for use in the fermented
feed of the present invention are naturally occurring and may be isolated
from suitable sources using techniques known in the art. Suitable sources
of lactic acid producing bacteria for use in the present invention
include the gastrointestinal tract of animals and birds, including but
not limited to the gastrointestinal tract of the target animal or bird of
the fermented feed concerned. Other sources of lactic acid producing
bacteria include cereal grains, spontaneous fermentations in substrates,
and the teats and other parts animals. Isolation of the lactic acid
producing bacteria may be carried out using techniques known in the art.

[0072] Lactic acid producing bacteria may be identified again using
techniques known in the art. For example, Lactobacilli may be identified
using the gram stain and catalase tests, with Lactobacilli being gram
positive and catalase negative rods.

[0073] In a further aspect, the present invention provides a method for
preparing a fermented feed composition, the method comprising fermenting
a feed substrate with a lactic acid producing bacteria, the lactic acid
bacteria being characterised by: [0074] a) being viable under the
conditions prevailing in the gastrointestinal tract of the target animal;
[0075] b) being an aggregating bacteria and/or co-aggregating with one or
more pathogenic bacteria; and [0076] c) being able to produce upon
fermentation in the feed substrate lactic acid in an amount of at least a
minimum inhibitory concentration of lactic acid.

[0077] The fermented feeds of the present invention may be prepared in any
suitable manner. Typically, the fermented feeds are prepared by
inoculating the feed substrate with an inoculum containing the lactic
acid producing bacteria in viable form and fermenting the feed substrate
under suitable conditions. Techniques for fermenting a feed substrate
after inoculation with a lactic acid producing bacteria are known in the
art.

[0078] The feed composition being fermented contains water. If a dry feed
substrate is being employed, water is added to the substrate. The feed
composition being fermented preferably contains water in an amount of
from 1 to 10 parts water by weight for each part of feed substrate (dry
basis), more preferably from 1 to 5 parts water, by weight. One preferred
embodiment comprises the feed substrate and water in a weight ratio of
from 1:1 to 1:3, more preferably from 1:1 to 1:2, especially from 1:1 to
1:1.5.

[0079] Fermentation of the feed substrate may be conducted at any
temperature suitable for the cultivation of the lactic acid producing
bacteria. The optimum temperature for fermentation will depend upon the
strain or strains of bacteria being employed. Typically, the feed
substrate is fermented at a temperature of from 15 to 45° C., more
preferably from 30 to 35° C.

[0080] The feed substrate is fermented for a sufficient period of time to
allow the lactic acid producing bacteria to produce at least a minimum
lactic acid concentration of 150 mMol/l lactic acid, more preferably at
least 200 mMol/l, still more preferably at least 250 mMol/l. Typical
fermentation times are from 8 to 72 hours, more preferably from 8 to 24
hours.

[0081] The production of lactic acid in the fermented feed may be
monitored by measuring the pH of the feed composition, which will fall as
lactic acid is produced during the fermentation process. The pH of the
feed composition after fermentation with the lactic acid producing
bacteria is preferably 4.5 or lower, more preferably 4.0 or lower.

[0082] As noted above, feeds having a low pH may be unpalatable to the
target animals. Fermented feeds having higher concentrations of lactic
acid and pH values below 3.5 may be advantageously combined with other
materials to produce a final diet, as long as the minimum inhibitory
concentration of lactic acid is maintained.

[0083] Nutrients and other components essential to the growth of the
lactic acid producing bacteria may be added to the feed substrate, as
required. Such nutrients and components will be known in the art.

[0084] The feed substrate is fermented to produce a concentration of
lactic acid producing bacteria in the feed composition that is beneficial
to the target animals. In particular, the lactic acid bacteria present in
the feed composition after fermentation is completed should be viable in
sufficient numbers to colonise the gastrointestinal tract of the target
animal and form viable colonies therein. Preferably the concentration of
lactic acid producing bacteria in the fermented feed is at least 106
CFU/ml, more preferably from 107 to 1010 CFU/ml, still more
preferably from 109 to 1010 CFU/ml.

[0085] The feed composition and method of the present invention may employ
any suitable lactic acid producing bacteria, with the proviso that the
bacteria is not harmful to the target animal. Preferred lactic acid
producing bacteria include strains of Lactobacillus and Pediococcus, with
strains of Lactobacillus being particularly preferred. Particularly
preferred microorganisms of the strain Lactobacillus include strains of
Lactobacillus plantarum and Lactobacillus salivarius.

[0086] Extensive work has been carried out to isolate a series of strains
of Lactobacillus of particular advantage in the preparation of fermented
feeds according to the present invention. The strains were isolated by
the general method described hereinbefore and using the detailed method
described below. Each of the isolated strains exhibited all three of the
properties (a) to (c) described above, making them particularly suitable
for use in the preparation of a feed composition according to the present
invention. Each of the isolated strains has been deposited on 11 Feb.,
2009, with the National Collections of Industrial and Marine Bacteria
Ltd., Aberdeen, Scotland (hereafter `NCIMB`) and accorded the NCIMB
accession numbers set out below. The deposits have been made pursuant to
and in satisfaction of the requirements of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Protection.

[0087] Accordingly, in a further aspect, the present invention provides
biologically pure cultures of the following microorganisms:

[0094] In a further aspect, the present invention provides a composition
for the preparation of a fermented feed, the composition comprising one
or more of the aforementioned microorganisms and a suitable carrier.

[0095] On a more general note, it has been found that the administration
to target animals of lactic acid producing bacteria having the following
characteristics: [0096] a) being viable under the conditions prevailing
in the gastrointestinal tract of the target animal; [0097] b) being an
aggregating bacteria and/or co-aggregating with one or more pathogens;
and [0098] c) being able to produce upon fermentation in the feed
substrate lactic acid in an amount of at least a minimum inhibitory
concentration of lactic acid;

[0099] is generally advantageous for the health and wellbeing of the
target animal.

[0100] Accordingly, in a further aspect, the present invention provides a
method for improving the general health of a target animal, the method
comprising administering to the animal lactic acid producing bacteria
having the following characteristics: [0101] a) being viable under the
conditions prevailing in the gastrointestinal tract of the target animal;
[0102] b) being an aggregating bacteria and/or co-aggregating with one or
more pathogens; and [0103] c) being able to produce upon fermentation in
the feed substrate lactic acid in an amount of at least a minimum
inhibitory concentration of lactic acid.

[0104] The microorganisms may be administered by way of the water provided
to the animals, for example as a single dose or by continuous feeding
with water containing the microorganisms. Alternatively, the
microorganisms may be administered by way of the feed provided to the
target animals, most preferably by way of a fermented feed as
hereinbefore described.

[0105] The preferred lactic acid producing bacteria for general
administering to target animals are as set out above.

[0106] The lactic acid producing bacteria are preferably administered to
the target animal as viable microorganisms, preferably in a concentration
of at least 106 CFU/ml, more preferably at least 107 CFU/ml,
still more preferably in a concentration of at least 109 CFU/ml. If
administered to the target animal by way of its water, the minimum number
of microorganisms is preferably at least 106 CFU/ml. If administered
by way of a fermented feed, the minimum number of lactic acid producing
bacteria is preferably at least 108 CFU/ml, more preferably up to
1010 CFU/ml.

[0107] It has been found that providing the target animals with lactic
acid producing bacteria in this way increases the rate at which the
animal increases in weight, and improves the overall health of the
animal, in particular increasing the resistance of the animal to
infection from potentially harmful microorganisms. This reduces the level
at which the target animals shed harmful bacteria into their environment,
in turn reducing the rate of infection of other animals in the vicinity
of the target animals. These advantages have been found to be
particularly marked when the lactic acid producing bacteria are provided
to very young or immature animals.

[0108] Further, the present invention provides a biologically pure culture
of a lactic acid producing bacteria having the following characteristics:
[0109] a) being viable under the conditions prevailing in the
gastrointestinal tract of the target animal; [0110] b) being an
aggregating bacteria and/or co-aggregating with one or more pathogens;
and [0111] c) being able to produce upon fermentation in the feed
substrate lactic acid in an amount of at least a minimum inhibitory
concentration of lactic acid.

[0112] A composition for providing lactic acid bacteria to a target animal
comprises a viable culture of the aforementioned lactic acid producing
bacteria having characteristics (a) to (c) and a suitable carrier.

[0113] The present invention will be illustrated by way of the following
specific examples and by reference to the accompanying figures, in which:

[0114] FIG. 1 is a diagrammatical representation of a preferred in vitro
method of determining the viability of lactic acid producing bacteria in
the gastrointestinal tract of the target animal;

[0115]FIG. 2 is a diagrammatical representation of a preferred method for
determining the aggregating and coaggregating ability of lactic acid
producing bacteria; and

[0116]FIG. 3 is a diagrammatical representation of a preferred method for
determining the antagonistic level of lactic acid producing bacteria.

[0117] The experiments described in the following examples were conducted
using lactic acid producing bacteria sourced, isolated and identified as
follows:

[0118] Three chickens (Hubbard breed; age 9 weeks and 2 days) were fed ad
libidum on a diet of a commercially available organic growers ration,
grass and clover. The chickens were humanely slaughtered and the entire
gastrointestinal tract removed from each bird. Contents from the caecum,
jejunum, ileum and crop were removed aseptically. In addition, epithelial
cells were removed from the small intestine and the crop by scraping with
a slide. All samples were diluted in 10 ml phosphate-buffered saline
(PBS, ex. Oxoid, England) and plated in Man-Rogosa-Sharpe (MRS) and
Rogosa agar (both ex. Oxoid, England). The streak method of isolation was
used to obtain pure cultures from a mixed culture of bacteria. The thus
isolated pure cultures were cultured for a second time in MRS agar plates
and incubated in anaerobic jars in an atmosphere containing 5% vol carbon
dioxide for 72 hours.

[0119] A total of 111 lactic acid producing bacteria were isolated on MRS
agar (isolation medium for Lactobacillus and Pediococcus strains) and
Rogosa agar (isolation medium for Lactobacillus strains).

[0120] Gram stains and catalase tests were used to confirm that the
isolates were lactic acid producing bacteria. Isolates that were Gram
positive and catalase negative were further identified by differential
carbohydrate metabolism using API CHL kits (ex. BioMereux, UK).

[0121] The lactic acid producing bacteria thus isolated and identified as
such were subjected to analysis using the procedures of Examples 1 to 3
to identify those meeting the requirements of: [0122] a) being viable
under the conditions prevailing in the gastrointestinal tract of the
target animal; [0123] b) being an aggregating bacteria and/or
co-aggregating with one or more pathogens; and [0124] c) being able to
produce upon fermentation in the feed substrate lactic acid in an amount
of at least a minimum inhibitory concentration of lactic acid.

[0125] In addition, the antagonistic activity of the isolated lactic acid
producing bacteria against key pathogenic bacteria was determined
following the procedure set out in Example 4. Further, the ability of the
bacteria to adhere to epithelial cells was determined using the procedure
set out in Example 5.

EXAMPLES

Example 1

Determination of Viability of Lactic Acid Producing Bacteria in the
Gastrointestinal Tract of the Target Animal

[0126] The viability of strains of lactic acid producing bacteria in the
gastrointestinal tract of chickens was determined using the following
procedure, which is summarised in FIG. 1:

[0127] Each strain of lactic acid producing bacteria was sprayed onto a
commercially available pelleted poultry grower feed (ex. Mole Valley
Farmers, Devon, UK). Prior to spraying with the bacteria, the feed was
sterilised by irradiation (25 kGy, Co60). The composition of the
pelleted feed was as follows:

[0128] A sample of the inoculated feed was added to a flask, diluted with
the addition of distilled water and heated in a water bath to
41.4° C., to represent the temperature within the gastrointestinal
tract of a chicken. The pH of the sample of the feed composition was
adjusted successively by the addition of HCl (aq; 1M) to adjust the pH in
the flask to correspond to the pH found at the successive stages in the
digestive tract of poultry: pH 4.4 to 4.5 to correspond to the crop and
proventiculus; pH 2.6 to correspond to the gizzard; and pH 6.2
corresponding to the small intestine. The sample was incubated at each pH
for a period of time corresponding to the time digesta take to pass
through the corresponding portion of the gastrointestinal tract: 45
minutes for the crop and proventiculus; 90 minutes for the gizzard; and
90 minutes for the small intestine.

[0129] HCl (aq; 1M) was added periodically to each sample throughout the
incubation period, in order to maintain the pH at the appropriate level
and counteract the normal buffering action of the feed components.

[0130] Samples (1 ml) of the solution in the flask was removed immediately
before the pH was adjusted at each stage in the incubation, diluted with
sterile peptone water (9 ml) and 10 fold serial dilutions were prepared.
100 μl of each dilution were spread over MRS agar using aseptic
techniques and the plates incubated at 37° C. for 24 hours, after
which the plates were counted. The viability of the microorganisms was
calculated as the percent of organisms surviving passage through the
simulated GI tract.

Example 2

Determination of Lactic Acid Bacteria that are Aggregating and
Coaggregating

[0131] The ability of the lactic acid bacteria strains to aggregate and
form coaggregates with other bacteria was determined using the following
procedure, as illustrated in FIG. 2:

[0132] Lactic acid producing bacteria were grown overnight in MRS broth
(ex Oxoid) at 37° C. in an atmosphere of 5% vol carbon dioxide.
Thereafter, the cultures were centrifuged for 10 minutes at 10000 times
gravity and washed three times with sterile distilled water. The thus
washed material was resuspended in the same volume of phosphate-buffered
saline (PBS) at a concentration of 109 CFU/ml at a pH of 6.0 and
incubated at room temperature.

[0133] Autoaggregation was determined to occur when clearly visible,
sand-like particles were formed by the aggregated cells and gravitated to
the bottom of the tubes within 2 hours.

[0134] In addition, the ability of the lactic acid producing bacteria to
co-aggregate with other bacteria was determined by the following
procedure:

[0135] The lactic acid producing bacteria were grown at 37° C. in
MRS broth for 24 hours in an atmosphere containing 5% vol carbon dioxide.
Salmonella spp. and E. coli were grown at 37° C. in nutrient for
24 hours in an atmosphere containing 5% vol carbon dioxide. Further,
Clostridium perfringens were grown in clostridial broth for 24 hours
under anaerobic conditions at 37° C. The following day, each
culture was centrifuged for 10 minutes at 10000 times gravity and washed
three times with sterile distilled water. The pathogenic cultures were
resuspended in phosphate-buffered saline (PBS) to the same initial volume
at a concentration of 109 CFU/ml (ph 6.0) and incubated at room
temperature in the presence of 10% vol freshly prepared filter-sterilised
culture of the lactic acid producing bacteria supernatant liquid, at a
total liquid volume of 1 ml. Coaggregation was taken as positive when
clearly visible, sand-like particles formed by aggregated cells settled
to the bottom of the vessel under gravity, leaving a clear supernatant
liquid within a period of 2 hours.

[0136] Co-aggregation of the lactic acid producing bacteria with other
potentially pathogenic microorganisms was tested in similar manner and
confirmed using scanning electron microscopy.

[0139] The antagonistic activity of the lactic acid producing bacteria
strains with respect to pathogenic microorganisms was determined using
the following procedure, as illustrated in FIG. 3:

[0140] The lactic acid producing bacteria were grown in MRS broth (ex.
Oxoid, CM0359) at a temperature of 37° C. for 24 hours under
anaerobic conditions. At the end of this period, samples of the bacteria
were spotted onto MRS agar plates (ex. Oxoid) using a sterile cotton swab
and incubated at 37° C. for a further 24 hours, again under
anaerobic conditions, to allow colonies to develop. Nutrient agar
containing approximately 107 CFU/ml of each of five pathogenic
bacteria Salmonella enteric Enteritidis (3 strains); Salmonella enteric
Typhimurium (1 strain); and Escherichia coli (1 strain) was poured on the
agar plate and the plate incubated for a further 24 hours at a
temperature of 37° C. Nutrient agar containing approximately
107 CFU/ml of Clostridium perfringens (1 strain) was poured on a
second plate and incubated under anaerobic conditions at 39° C.
for a further 48 hours.

[0141] At the end of the incubation periods, the plates were checked
visually for inhibition zones around the Lactobacilli spots and the
radius of the inhibition zone was recorded. A measure of the antagonistic
activity of the subject lactic acid producing bacteria against the target
pathogens may be obtained by measuring the radius of the inhibition zone
around the lactic acid producing bacteria spot. A radius of from 1 to 2
cm indicated a high level of antagonistic activity.

[0142] An experiment was conducted to determine the ability of strains of
lactic acid producing bacteria to adhere to epithelial cells of
organically farmed chickens using the following procedure.

[0143] The chickens were humanely slaughtered and ileal epithelial cells
were removed by scraping the epithelium with a microscope slide. The
cells thus removed were suspended in PBS and examined to ensure that they
were free from any adherent bacteria. A haemocytometer was used to
determine the number of cells.

[0144] Selected Lactobacilli were cultured overnight in MRS broth to give
bacterial count of 109 CFU/ml, and resuspended in PBS to give a cell
density of 108 CFU/ml. 100 μl of the Lactobacillus suspension was
added to 400 μl of the epithelial cell suspension and the mixture
incubated for 30 minutes at 37° C. with shaking. Adhesion of the
Lactobacillus cells to the epithelial cells was observed using a phase
contrast microscope by counting the number of bacterial cells adhered to
epithelial cells selected at random from the resulting suspension.

Example 6

[0145] An experiment was conducted to determine the benefits of treating
chickens with strains of the lactic acid-producing microorganism
Lactobacillus salivarius exhibiting the characteristics of a) being
viable in the gastrointestinal tract of chickens (determined as outlined
in Example 1); b) aggregating and co-aggregating with at least one or the
following pathogens: Salmonella, E. coli, or Clostridia (determined as
outlined in Example 2); and c) producing at least a minimum inhibitory
concentration of lactic acid (determined as outlined in Example 3). In
addition, the microorganisms were determined to be antagonistic to
strains of Salmonella, Clostridium and E. coli using the method set out
in Example 4. Using the procedure set out in Example 5, the lactic
acid-producing bacteria were also determined to be highly adherent to
chicken epithelial cells.

[0146] The strain of Lactobacillus salivarius employed was strain C28
referred to above.

[0147] Throughout the experiment, the birds were fed on a diet of clean
water and a commercially available feed (Saracen Chick Crumbs, ex. J&W
Attlee, Dorking, England). The feed had a moisture content of 14.0 wt %,
with the composition, on a dry basis, as set out in Table II.

[0148] 102 specific pathogen-free chickens were randomly allotted to six
groups of 17, with each group being treated as follows:

[0149] Group I: birds fed clean water and feed according to Table I.

[0150] Group II: birds treated by oral gavage at age 1 day with an aqueous
medium containing Lactobacillus salivarius in a concentration of 107
cfu/ml. Thereafter, the birds were fed as for Group I.

[0151] Group III: birds fed water containing 107 cfu/ml Lactobacillus
salivarius and feed according to Table I from age 1 day.

[0152] Group IV: birds fed water containing 107 cfu/ml Lactobacillus
salivarius and feed according to Table I from age 7 days.

[0153] Group V: birds fed clean water and fermented wet mash from age 1
day. The fermented wet mash was prepared by inoculating a mixture of the
feed of Table I and water (ratio of feed to water of 1:1.2) with
Lactobacillus salivarius and fermenting for 24 hours at 30° C. to
obtain a microorganism concentration of about 109 cfu/nil. The feed
had a mean pH before fermentation with Lactobacillus salivarius of 5.94
and a mean pH after fermentation of 4.42.

[0154] Group VI: birds fed clean water and fermented wet mash from age 7
days. The fermented wet mash was prepared by inoculating a mixture of the
feed of Table I and water (ratio of feed to water of 1:1.2) with
Lactobacillus salivarius and fermenting for 24 hours at 30° C. to
obtain a microorganism concentration of about 109 cfu/ml. The feed
had a mean pH before fermentation with Lactobacillus salivarius of 5.94
and a mean pH after fermentation of 4.39.

[0155] Weight of Feed Consumed by Birds

[0156] The experiment was conducted for a period of six weeks. As a first
indicator of the health of the birds, the weight of feed being consumed
by the birds in each group was monitored. The average daily feed
consumption of the birds in each group during weeks 3 to 6 of the
experiment is set out in Table 2.

[0157] As shown in Table 2, birds in Groups V and VI provided with the
fermented feeds consumed significantly higher quantities of feed than the
birds in other groups, indicative of a higher general level of health for
the birds in Groups V and VI. In general, the birds provided with the
lactic acid producing bacteria, whether by way of water or fermented
feed, consumed larger quantities of feed, compared with the control group
I.

[0158] Weight Gained by Birds

[0159] In addition to the quantity of feed consumed, the average weight
gain for the birds in each group was measured. The average daily weight
gain of the birds in weeks 3 to 5 of the experiment is shown in Table 3.

[0160] As shown in Table 3, the average daily weight gain of the birds
treated with Lactobacillus salivarius was generally higher than that of
the untreated birds. In particular, the initial weight gain of all birds
provided with the lactic acid producing bacteria was significantly higher
than that of the control group I. Further, the birds in Groups V and VI
fed on fermented feed exhibited significantly higher weight gain, in
particular in week 5 of the experiment.

[0161] Salmonella Shedding and Infection of Birds

[0162] At the start of the experiment, a random sample of birds from each
group was cloacally swabbed and their faecal contents analysed to confirm
no infection by strains of Salmonella.

[0163] To determine the effectiveness of the treatment with Lactobacillus
salivarius in preventing infection of the birds by other microorganisms,
all birds in each group were challenged with a strain of Salmonella
according to the following procedure:

[0164] At 15 days of age, all birds were dosed with Salmonella typhimurium
by oral gavage using a dosing catheter to administer an aqueous medium
containing 106 cfu/ml of Salmonella microorganisms. The Salmonella
organisms employed were a nalidixic acid resistant derivative (SL1344
nalr; ex. Veterinary Laboratories Agency (VLA), Weybridge, UK).
Immediately prior to dosing with Salmonella, the birds were dosed in like
manner with a solution of sodium bicarbonate, so as to instantaneously
neutralise the acidity in the crop of the bird. In this way, the barrier
imposed in the upper gut of the birds by acids in the crop was removed,
permitting access of the introduced Salmonella to the lower gut
environment.

[0165] Cloacal swabs were taken from the birds immediately before
challenge and at least twice a week after challenge for a period of 4
weeks. The content of Salmonella typhimurium in the swabbed material was
determined. In addition, the Salmonella-content of the bird litter was
determined for each group. For each group, the percentage of birds that
were found not to be shedding salmonella was determined. The results are
set out in Table IV.

[0166] From Table IV it can be seen that, in general, administering lactic
acid producing bacteria to the birds significantly reduced the tendency
of the birds to shed Salmonella into their environment. The reduction in
Salmonella of the birds provided with the fermented feed, that is Groups
V and VI, is particularly marked.

[0167] Further, throughout the duration of the experiment, it was found
that at all times, the Salmonella shedding exhibited by the birds was
consistently and significantly lower in the groups fed with the fermented
feeds, that is Groups V and VI.

[0168] To determine the level of infection of the birds in each group, two
post-mortem enumerations of Salmonella typhimurium infestations of the
birds were carried out, one at 4 weeks of age and one at 6 weeks of age.
The method employed was as follows:

[0169] The birds were euthanized by cervical dislocation. Their liver,
spleen, ileum and caeca were removed aseptically and placed in sterile
PBS. Samples of each tissue thus collected were weighed, homogenised and
subjected to serial 10 fold dilutions in PBS (0.1M; pH 7.2). The viable
count of Salmonella typhimurium in each homogenate was determined by
plating drops of the dilutions on BGA supplemented with nalidixic acid
(15 μg/ml). 1.0 ml of residual homogenate was added to 10 ml Selenite
enrichment broth, incubated for 24 hours at 37° C., and thereafter
subcultured on BGA supplemented with nalidixic acid. Viable counts of
bacteria were determined by plating on MRS agar and incubating in
anaerobic jars for 48 hours at 37° C.

[0170] The results of the first and second post mortem tests are set out
in Tables V and VI respectively.

[0171] From Tables V and VI it can be seen that, in general, providing the
birds with lactic acid producing bacteria significantly reduced the count
of Salmonella typhimuriam in the birds. The reduction in the count of
Salmonella typhimuriam was most notable in the birds in Groups V and VI
fed with the fermented feeds.

[0172] Lactobacillus Salivarius Colonisation of Birds

[0173] For the birds euthanized in the tests described above, the count of
Lactobacillus salivarius in the caecum and ileum of the birds was also
determined, using the general procedure outlined above, in order to
determine the efficiency of the lactic acid producing bacteria in
colonising the gastrointestinal tracts of the birds. The results are set
out in Table VII.

[0174] As shown in Table VII, the provision of the lactic acid producing
bacteria to the birds in the water and the feed significantly increased
the degree of colonisation of the gastrointestinal tract by the bacteria.
The fermented feeds provided to the birds in Groups V and VI were
particularly effective in increasing the concentration of lactic acid
producing bacteria in the gastrointestinal tract of the birds.

[0175] As a general result, the fermented feeds were particularly
effective in reducing the colonisation of the birds by Salmonella. This
is of significant advantage in the production of foodstuffs for humans,
were the prime concern of food producers is the elimination of food bore
pathogens, such as Salmonella from food animals and their products.
Surprisingly, it was found that improved resistance to colonisation with
Salmonella was achieved by providing the birds with fermented feed only
from the age of 7 days, and not from age 1 day.